Bok-Kyu Shin

Absolute configurations of isoflavan-4-ol stereoisomers

Isoflavan-4-ol has been synthesized quantitatively from the reduction of isoflavone in the presence of Pd/C and ammonium formate under N(2) atmosphere. Isolation of cis- and trans-isomers was achieved by flash column chromatography and each enantiomer was separated by Sumi-Chiral column chromatography. Absolute configurations of four stereoisomers were determined by circular dichroism spectroscopy.

Anti-obesity Effect of Carbon Dioxide Supercritical Fluid Extracts of Panax Ginseng C. A. Meyer

The root of Korean ginseng, Panax ginseng C. A. Meyer, has been known worldwide as a panacea for many centuries. Contradictory weight gain and loss effects in Korean ginseng led to the adoption of supercritical carbon dioxide extraction to assure the quality of food-grade anti-obesity ginseng products. Among different extraction conditions of CO2 pressure (200 and 300 bars) and temperature (40, 50, and 60°C), the highest extraction yield of 0.66% was obtained with 300 bars at 40°C. To test anti-obesity effect, each supercritical fluid extract (SFE) was supplemented to the high-fat diet of male obese-prone C57BL/6J strain mice. All carbon dioxide SFEs of ginseng showed significant weight-loss effects in the tested mice. The average weights of normal and high fat diet mice were 28.7±2.0 and 35.0±1.6 g, respectively, after 10 weeks. Average weights of mice on SFE-supplemented high fat diets were between 24.5±1.4 and 25.9±1.3 g. SFEs obtained at 50°C, and at both 200 and 300 bars, showed the most profound anti-obesity effect. Both SFEs showed ~15 and ~30% weight loss for the normal and high-fat diet mice, respectively. The major component of the SFE was analyzed as polyacetylene compounds, and panaxydol and panaxynol were identified as major polyacetylene compounds. However, the anti-obesity effect of the ginseng SFEs was not correlated with contents of these two polyacetylene compounds in SFEs. No side-effects of ginseng SFEs were observed, and the anti-obesity effect was reversible.

N-Terminal Region of GbIspH1, Ginkgo biloba IspH Type 1, May Be Involved in the pH-Dependent Regulation of Enzyme Activity

GbIspH1, IspH type 1 in Ginkgo biloba chloroplast, is the Fe/S enzyme catalyzing the reductive dehydroxylation of HMBPP to isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) at the final step of methylerythritol phosphate pathway in chloroplast. Compared to the bacterial IspH, plant IspH, including GbIspH1, has an additional polypeptide chain at the N-terminus. Here, biochemical function of the N-terminal region of GbIspH1 was investigated with the N-terminal truncated GbIspH1 (GbIspH1-truncated). Both wild type GbIspH1 (GbIspH1-full) and GbIspH1-truncated were catalytically active and produced IPP and DMAPP in a ratio of 15u2009:u20091. Kinetic parameters of K M (17.3 ± 1.9 and 14.9 ± 2.3u2009µM) and k cat (369 ± 10 and 347 ± 12u2009min−1) at pH 8.0 were obtained for GbIspH1-full and GbIspH1-truncated, respectively. Interestingly, GbIspH1-full and GbIspH1-truncated showed significantly different pH-dependent activities, and the maximum enzyme activities were obtained at pH 8.0 and 7.5, respectively. However, catalytic activation energies (E a) of GbIspH1-full and GbIspH1-truncated were almost the same with 36.5 ± 1.6 and 35.0 ± 1.9u2009kJ/mol, respectively. It was suggested that the N-terminal region of GbIspH1 is involved in the pH-dependent regulation of enzyme activity during photosynthesis.

Characterization of Burkholderia glumae BGR1 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (HDR), the terminal enzyme in 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway

Abstract4-Hydroxy-3-methylbut-2-enyl diphosphate reductase (HDR) is the ultimate enzyme in 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway converting (E)-4-hydroxy-3-methylbut-2-enyl pyrophosphate (HMBPP) into isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Burkholderia glumae, a Gram-negative rice-pathogenic bacterium, harbors 2 hdr genes and lacks isopentenyl diphosphate isomerase (idi). Both HDR enzymes could complement E. coli hdr deletion mutant (DYTL1). Both of the recombinant HDR proteins, BgHDR1 and BgHDR2, catalyzed reduction of HMBPP into IPP and DMAPP at a ratio of 2:1, in contrast to 5:1 ratio of other bacterial HDRs so far characterized. The kcat and Km values of BgHDR1 and BgHDR2 were 187.0 min−1 and 6.0 μM and 66.6 min−1 and 21.2 μM, respectively. Physiological significance of the kinetic properties was discussed.

High-throughput HDR inhibitor screening

Isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), biochemical precursors of the isoprenoids, are biosynthesized by mevalonate pathway and methylerythritol phosphate (MEP) pathway. Because these two pathways are mutually exclusive in most organisms, inhibition of MEP pathway has become a target for the development of new bioactive materials, including antibiotics, antimalarial drugs, and herbicides. In the final step of MEP pathway, (E)-4-hydroxy-3-methylbut-2-enyl diphosphate reductase (HMBPP reductase, HDR) catalyzes reduction of HMBPP to IPP and DMAPP. HDR requires electron transfers to the Fe/S cluster in the active site for the catalysis, and methyl viologen has been used as a common redox mediator for in vitro studies. We developed a high throughput colorimetric HDR inhibitor screening method by utilizing microtiter-plate screening method. This method was applied to various plant extracts to measure HDR inhibition activity, and potent HDR inhibitor was successfully screened.

Exceptionally high percentage of IPP synthesis by Ginkgo biloba IspH is mainly due to Phe residue in the active site

(E)-4-Hydroxy-3-methylbut-2-enyl diphosphate (HMBPP) reductase (IspH, HDR or LytB) is an Fe/S enzyme catalyzing the reductive dehydroxylation of HMBPP to isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) in the last step of methylerythritol phosphate (MEP) pathway. The MEP pathway is known to produce 4-6:1 ratio of IPP and DMAPP mixture by the last enzyme, IspH. Plant IspH in plastids follows same catalytic mechanism as others, but GbIspH (Ginkgo biloba IspH) was reported to produce a mixture of IPP and DMAPP in a ratio of 16:1. Present catalytic mechanisms of IspH involve a common allyl anion intermediate, and the intramolecular proton transfer to the allyl moiety is considered as the key reaction step determining the product between IPP and DMAPP. The F212 residue in plant IspH was found as a potential amino acid residue that could mediate the proton transfer to the allyl anion intermediate before the product release. In this report, catalytic function of GbIspH F212 residue (H74 in E.xa0coli), especially during the product formation in the active site, was studied by means of site-directed mutation. The product ratio of IPP/DMAPP was measured as 6.5xa0±xa00.1 for F212H GbIspH, and the value was close to the reported bacterial IspH having His residue on that specific position. Along with the other F212Y mutant, of which ratio was determined as 10.9xa0±xa00.1, the results strongly support that the Phe residue in plant IspH is the key amino acid residue that allows exclusive production of IPP in plant chloroplast.